Our long-term goal for this project is to advance our understanding of developmental disorders of the brainstem and cerebellum, brain structures derived from the embryonic midbrain and hindbrain, which we refer to collectively as mid-hindbrain malformations (MHM). These disorders affect a minimum of 1 per 6-7000 live births, and likely far more as these numbers do not account for cerebellar abnormalities associated with preterm birth or with autism. Further, cerebellar malformations are known to co-occur with several more common developmental disorders including autism, mental retardation and some types of early life epilepsy. With this renewal, we propose to continue using our large and growing cohort of human subjects with MHM to define the genes, pathways and biological mechanisms underlying these developmental disorders. We will use the most recent genomic technology - massively parallel (NextGen) sequencing of targeted gene panels, whole exome sequencing (WXS) or whole genome sequencing (WGS) - combined with older methods to find the causes of both rare and common MHM. In Aim 1, we will continue to search for genes underlying rare single gene causes of MHM that will demonstrate the most important molecular pathways, including pathways that contribute to more common disorders such as autism. As an example, we have identified the first missense mutation of the AUTS2 gene (previously linked to autism) in a child with MHM. In Aim 2, we will turn to the more challenging but also more important problem of Dandy-Walker malformation, the most common MHM in humans. This specific malformation demonstrates substantial causal heterogeneity and has proven difficult to solve with older technologies, making whole exome and genome sequencing approaches essential. Aims 1-2 need to be supported by ongoing subject recruitment, as studies of comparable disorders such as mental retardation and autism have benefited from large numbers of subjects. In Aim 3, we propose to test the biological function of genes and networks identified in Aims 1-2 using new CRISPR/Cas technology to efficiently generate new mouse models of proven and strong candidate MHM-associated genes. For example, we are now generating the first mouse models of Auts2. We expect that these studies will contribute rapidly to more accurate diagnosis and counseling, and over time will lead to development of specific treatments for a subset of these disorders. We predict that studies of mid-hindbrain development will have broad significance for human developmental disorders generally, providing compelling evidence for a connection between cerebellar development and other classes of developmental disorders.